The common endocrine disorder of primary hyperparathyroidism (PHPT) is caused by a disruption of appropriate calcium sensing in the parathyroid glands. The disease is characterized by constitutively elevated secretion of the parathyroid hormone (PTH) associated with neoplastic transformation of one (single adenoma), two (double adenoma), or all four (hyperplasia) of the parathyroid glands. Approximately 85% of PHPT results from a single adenoma. The prevailing view maintains that clonal expansion of a parathyroid tumor progenitor cell harboring an initiating genomic lesion coupled with transcriptional down-regulation of the calcium sensing receptor (CASR) in the resulting culprit gland(s) instigates the loss of calcium responsiveness in PHPT. Although many parathyroid tumors arise clonally, new data from our laboratory supports an alternative etiological model in which attenuated calcium sensitivity leads to an abnormal calcium/PTH relationship, driving polyclonal proliferation of parathyroid cells in response to chronic demand for increased PTH. Using a preparative flow cytometry approach, we analyzed the composition of human parathyroid adenomas and found these tumors to be comprised of functionally and morphologically distinct cellular subpopulations that are highly heterogeneous with respect to calcium responsiveness, CASR expression, and clonal origin. Moreover, in human tissues and in a transgenic mouse model developed in our laboratory, we and others have found that multiple mechanisms including altered subcellular trafficking of CASR and increased biochemical antagonism via downstream modifiers such as the RGS5 protein, Ga11, AP2S1 can contribute to calcium desensitization and neoplastic transformation in the parathyroid gland. In particular, we have observed striking regional variations in CASR signaling, localization, and abundance within individual parathyroid adenomas. These important new indicators of parathyroid intratumoral heterogeneity reveal a previously unappreciated degree of complexity in the etiopathogenesis and clinical diversity of PHPT. We propose to build upon these provocative results by performing functional assessments of human parathyroid neoplastic tissue at single cell resolution in order to define the precise determinants of calcium responsiveness and elucidate how these molecular indices relate to parathyroid disease etiology, clinical presentation, and outcome.